EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 19 to 23 October 2009

Transcription

1 ENGLISH ONLY FINAL EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 19 to 23 October 2009 Recommendations to assure the quality, safety and efficacy of pneumococcal conjugate vaccines Replacement of: TRS 927, Annex 2. World Health Organization 2009 All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: ; fax: ; Requests for permission to reproduce or translate WHO publications whether for sale or for noncommercial distribution should be addressed to WHO Press, at the above address (fax: ; e- mail: The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors [or editors as appropriate] alone are responsible for the views expressed in this publication. Adopted by the 60 th meeting of the WHO Expert Committee on Biological Standardization, 19 to 23 October A definitive version of this document, which will differ from this version in editorial but not scientific details, will be published in the WHO Technical Report Series.

2 Page 2 Recommendations published by the WHO are intended to be scientific and advisory. Each of the following sections constitutes guidance for National Regulatory Authorities (NRAs) and for manufacturers of biological products. If a National Regulatory Authority (NRA) so desires, these Recommendations may be adopted as definitive national requirements, or modifications may be justified and made by the NRA. It is recommended that modifications to these Recommendations be made only on condition that modifications ensure that the vaccine is at least as safe and efficacious as that prepared in accordance with the recommendations set out below. The parts of each section printed in small type are comments for additional guidance intended for manufacturers and NRAs, which may benefit from those details. Introduction...3 General considerations...3 Part A. Manufacturing recommendations...6 A.1 Definitions...6 A.2 General manufacturing requirements...7 A.3 Production Control...8 A.4 Records...21 A.5 Retained samples...21 A.6 Labelling...21 A.7 Distribution and transport...21 A.8 Stability, storage and expiry date...21 Part B. Non-clinical evaluation of new pneumococcal conjugate vaccines...23 Part C. Clinical evaluation of pneumococcal conjugate vaccines...24 C.1 Considerations for clinical studies...24 C.2 Assessment of immune responses...25 C.3 Other possible indications for use...32 C.4 Post-marketing studies of safety and effectiveness...32 Part D. Recommendations for national regulatory authorities...33 D.1 General...33 D.2 Release and certification...33 D.3 Consistency of manufacture...33 Acknowledgements...34 References...36 Appendix...41

3 Introduction Page 3 This guideline provides recommendations for the production and control of pneumococcal conjugate vaccines in Part A and for non-clinical evaluation in Part B. Part C considers the content of the clinical development program applicable to pneumococcal conjugate vaccines that are primarily intended for the prevention of invasive pneumococcal disease (IPD) and for administration to infants and toddlers. The clinical assessment of the potential for these vaccines to prevent IPD in older children and adults (including the elderly) or to prevent other types of pneumococcal infection (e.g., pneumonia and otitis media) is not considered in any detail. General considerations Infections caused by Streptococcus pneumoniae are responsible for substantial morbidity and mortality, particularly in the very young and in the elderly (1-3). Pneumococci are grouped into many serotypes (~ 91) on the basis of their chemically and serologically distinct capsular polysaccharides. Certain serotypes are much more likely than others to be associated with clinically apparent infections, to cause severe invasive infections and to acquire resistance to one or more classes of antibacterial agents (4) The capsular polysaccharides of 23 serotypes are included in licensed non-conjugated polysaccharide vaccines produced by various manufacturers. Non-conjugated pneumococcal polysaccharide vaccines elicit T-cell independent immune responses. As a result, they do not elicit protective immune responses in children aged less than approximately 2 years and they do not induce immune memory. In addition, they have little or no impact on nasopharyngeal carriage (5). However, they are widely recommended for use in the elderly and in subjects from the age of approximately two years who have underlying medical conditions that put them at high risk of developing IPD (6). The development of pneumococcal conjugate vaccines, in which each of the selected bacterial capsular polysaccharides is coupled with a protein carrier molecule, has been a major advance in the prevention of IPD (7-10). In contrast to the 23-valent non-conjugated vaccines, the conjugated vaccines induce T-cell dependent immunity. As a result, they are immunogenic in infants under two years of age and they elicit immune memory. Since 2006, WHO has recommended that all countries should incorporate pneumococcal conjugate vaccines in routine immunization schedules for children aged less than 2 years with prioritization of their introduction in countries with high child mortality rates and/or high rates of HIV infection (6). A 7-valent pneumococcal conjugate vaccine (7vPnC) that employs CRM197 as the carrier protein for all seven serotypes was the first to be developed. This vaccine was first licensed in the USA in 2000 and subsequently has become available in approximately 90 countries worldwide. Pneumococcal conjugate vaccines that contain three (11) or six serotypes in addition to those in the 7vPnC vaccine have recently become available in some countries. The 10-valent vaccine includes tetanus toxoid, diphtheria toxoid or a novel protein derived from non-typable Haemophilus influenzae (protein D) as the carrier proteins while the 13-valent vaccine uses only CRM197 as the carrier protein. Vaccine efficacy (VE) against IPD has been evaluated in randomized and controlled studies in children aged less than 2 years. The studies employed the 7vPnC vaccine or an experimental 9vPnC vaccine that included all seven serotypes in the 7vPnC vaccine. At the time that these studies were initiated, there was no licensed pneumococcal conjugate vaccine available; consequently, the control groups did not receive a pneumococcal conjugate vaccine. The studies provided data from the USA, (in the general population and in native American children) (7, 8, 12), South Africa (13) and The Gambia (14). The 7vPnC vaccine and the 9vPnC vaccine were

4 Page 4 shown to be efficacious in preventing IPD although serotype-specific efficacy could be estimated for only four of the serotypes. Post-marketing effectiveness data from countries in which the 7vPnC vaccine has been introduced into the routine infant and toddler immunization programs have shown a reduction in rates of IPD in children aged less than 2 years due to all seven vaccine serotypes and also due to serotype 6A that is not included in the vaccine (7, 15). In addition, routine use of the 7vPnC vaccine in infants and toddlers has been associated with reductions in rates of IPD in the elderly population, indicating that there is an indirect beneficial effect (i.e. a herd immunity effect) on unvaccinated persons (15). Correspondingly, studies have demonstrated that the 7vPnC vaccine reduces rates of nasopharyngeal carriage of serotypes included in the vaccine and some types that are not included. Thus far the safety profiles of 7vPnC vaccine is considered to be acceptable (9, 10, 16, 17). WHO recommendations for pneumococcal conjugate vaccine production and control were first established in 2003 and were published in the WHO Technical Report Series (TRS) 927, annex 2. In that document it was considered that practical or ethical considerations may make it impossible to perform protective efficacy trials i.e. using an unvaccinated control group. Therefore, the recommendations discussed the design of immunogenicity studies that should be performed to support the licensure of new pneumococcal conjugate vaccines (including those containing conjugated capsular polysaccharides of serotypes additional to those in the 7vPnC vaccine) intended to prevent IPD and for administration to children aged less than 2 years. It was considered essential that the immunogenicity studies with a new pneumococcal conjugate vaccine should provide a link back to the VE against IPD that was demonstrated for the 7vPnC vaccine. Therefore, it was recommended that immune responses to each serotype in the 7vPnC vaccine that is also included in a new pneumococcal conjugate vaccine should be directly compared in randomized clinical studies and that the primary comparison of immune responses should be based on serotype-specific IgG antibody concentrations measured by enzyme-linked immunosorbent assay (ELISA). In order to facilitate these comparisons a WHO reference ELISA assay was established that includes pre-adsorption of sera with pneumococcal C polysaccharide (C-PS) and serotype 22F polysaccharide. The Appendix explains these pre-adsorption steps and provides details of the validation, standardization and bridging of ELISA assays. The immunogenicity data and estimates of VE against IPD across all serotypes in the 7vPnC vaccine were pooled for three of the abovementioned randomized, controlled efficacy studies in infants and toddlers (see Table 1). IgG concentrations in sera were measured using a well characterized ELISA method that differed from the WHO reference ELISA only in that it did not include a 22F adsorption step. It was subsequently demonstrated, that for this particular ELISA protocol, the pre-absorption of sera with C-PS and 22F had a minimal effect on estimations of IgG concentrations in a selection of sera from infants who received 7vPnC or 9vPnC vaccines (18). Based on these data an IgG antibody concentration of 0.35 µg/ml (assessed using the WHO ELISA) was suggested for use as a benchmark (or threshold value) when comparing immune responses to each serotype common to the 7vPnC vaccine and a new pneumococcal conjugate vaccine (19). The rationale for selecting this threshold antibody concentration is described in more detail in the proceedings of a WHO meeting (20). Briefly, results from three clinical trials (Table 1) were pooled to derive the 0.35µg/ml threshold. The number of IPD cases in the vaccinated and unvaccinated cohorts of each trial as well as the total number of participants were summed and used to calculate a pooled estimate of 93% for the vaccine efficacy. This vaccine efficacy was then referred to a pooled reverse cumulative distribution (RCD) curve to infer the final 0.35 µg/ml threshold (18). Therefore, this value is not an average estimate using the trialspecific thresholds listed in table 1, it is derived from the pooled RCD curve.

5 Page 5 Table 1 Estimated antibody concentration threshold using immunogenicity and vaccine efficacy (VE) data from 3 clinical trials (18). Study Patients (per protocol) VE observed Estimated threshold Control PCV µg/ml 95% CI NCKP 10,995 (MnCC)* 10,940 (7vPCV) 97.4% 0.20 (0.03, 0.67) American Indian 2,818 (MnCC) 2,974 (7vPCV) 76.8% 1.00 (0.25 > 50.00) South Africa 18,550 (Placebo) 18,557 (9vPCV) 90% 0.68 (0.03, 6.00) Pooled (unweighted) 93% 0.35 (0.09, 0.89) Pooled (weighted) 93% 0.35 (0.11, 0.85) *MnCC = Meningococcal group C conjugate vaccine While this population-derived IgG antibody threshold value is considered to be a useful benchmark it is important that it is not interpreted to mean that achievement of 0.35 µg/ml for a specific serotype (whether included in the 7vPnC vaccine or in a new pneumococcal conjugate vaccines) predicts protection against IPD due to that serotype in an individual subject. It was recognized that a threshold based on opsonophagocytic assay (OPA) titers (which reflect functional antibody) might also be suitable for comparing immune responses between vaccines and it was recommended that OPA data should be generated for a subset of vaccinated subjects in clinical studies. The limited data obtained during the protective efficacy studies conducted with the 7vPnC vaccine indicated that an IgG concentration 0.2 µg/ml (determined using an ELISA without 22F pre-adsorption of sera) corresponded approximately to an OPA titer 1:8 for some serotypes (20). s for determining OPA are also discussed in the Appendix. Prompted by issues raised during the development of newer pneumococcal conjugate vaccines since the publication of TRS 927 annex 2 in 2003, the WHO held a consultation in 2008 (21) to consider new scientific evidence and to discuss the need to provide revised guidance for manufacturers and licensing authorities. For example, the consultation reviewed effectiveness data obtained with various immunization schedules of the 7vPnC vaccine in the USA (7), Canada (22) and UK (23) There was a consideration of technical developments in ELISA and OPA methods, variability between assays and the need for standardization. The importance of bridging assays to determine IgG concentrations to the WHO reference ELISA method was discussed along with the option of establishing an assay-specific alternative threshold value to 0.35 µg/ml. At the 2008 consultation meeting some data were provided that supported the use of the IgG antibody threshold as a benchmark value. For example, data from the UK had shown that only 30-50% of infants reached the threshold of 0.35 µg/ml against 6B after two doses of 7vPnC vaccine at 2 and 4 months of age and this was associated with vaccine failures due to 6B in the interval between the second dose and the third dose at 13 months. However, previous and newer data suggested that IgG antibody concentrations less than 0.35 µg/ml may be sufficient to prevent IPD due to some serotypes. In addition, some data suggested that OPA titers against certain serotypes (e.g. 19A) correlated better with estimates of effectiveness than IgG concentrations when measured using the WHO reference assay (24).

6 Page 6 Overall it was considered that some of the new information accrued since 2003 merited incorporation into updated WHO Recommendations for pneumococcal conjugate vaccines. The majority of the revisions pertain to the clinical assessment of new pneumococcal vaccines. Part A. Manufacturing recommendations A.1 Definitions A.1.1 Proper Name The proper name of the vaccine shall be "pneumococcal conjugate vaccine" translated into the language of the country of use. The serotypes included in the vaccine should be associated with the name of the vaccine and listed in the packaging material. The use of this proper name should be limited to vaccines that satisfy the requirements formulated below. A.1.2 Descriptive definition Multivalent pneumococcal conjugate vaccine is a preparation of capsular polysaccharide from specific serotypes of Streptococcus pneumoniae that are covalently linked to carrier protein. A.1.3 International Reference Materials No formally established international reference materials that would allow the standardization of immune responses to pneumococcal conjugate vaccines are currently available. The following reagents are available through the courtesy of individuals, manufacturers and national control or reference laboratories: C-polysaccharide (Statens Serum Institut, Copenhagen, Denmark) Capsular polysaccharides (ATCC, Manassas, Virginia, USA) 89-SF reference serum (CBER/FDA, Washington DC, USA) 96DG secondary reference serum (provided by Dr David Goldblatt and distributed by NIBSC, UK) ELISA calibration sera (provided by Dr David Goldblatt and distributed by NIBSC, UK) (Plikaytis et al 2000). Pneumococcal serotyping reagents (Statens Serum Institut, Copenhagen, Denmark). HL-60 cells (ATCC, Manassas, Virginia, USA or ECACC, Porton Down Salisbury, UK) A.1.4 Terminology Master seed lot. A bacterial suspension of S. pneumoniae derived from a strain that has been processed as a single lot and is of uniform composition. It is used for the preparation of the

7 Page 7 working seed lots. Master seed lots shall be maintained in the freeze dried form or be frozen below -45 C. Working seed lot. A quantity of live S. pneumoniae organisms derived from the master seed lot by growing the organisms and maintaining them in aliquots in the freeze-dried form or frozen state at or below -45 C. The working seed lot is used, when applicable, after a fixed number of passages, for the inoculation of production medium. Single harvest. The material obtained from one batch of cultures that have been inoculated with the working seed lot (or with the inoculum derived from it), harvested and processed together. Purified polysaccharide. The material obtained after final purification. The lot of purified polysaccharide may be derived from a single harvest or a pool of single harvests processed together. Modified polysaccharide. Purified polysaccharide that has been modified by chemical reaction or physical process in preparation for conjugation to the carrier. Carrier. The protein to which the polysaccharide is covalently linked for the purpose of eliciting a T-cell dependent immune response to the pneumococcal polysaccharide. Monovalent Bulk Conjugate. A conjugate prepared from a single lot or pool of lots of polysaccharide and a single lot or a pool of lots of protein. This is the parent material from which the final bulk is prepared. Final Bulk Conjugate. The blend of monovalent conjugates present in a single container from which the final containers are filled, either directly or through one or more intermediate containers derived from the initial single container. Final Lot. A number of sealed, final containers that are equivalent with respect to the risk of contamination during filling and, when it is performed, freeze-drying. A final lot must therefore have been filled from a single container and freeze-dried in one continuous working session. A.2 General manufacturing requirements The general manufacturing recommendations contained in Good Manufacturing Practices for Pharmaceuticals (25) and Biological Products (26) should be applied to establishments manufacturing pneumococcal conjugate vaccines with the addition of the following: Details of standard operating procedures for the preparation and testing of pneumococcal conjugate vaccines adopted by the manufacturer together with evidence of appropriate validation of each production step should be submitted for the approval of the NRA. All assay procedures used for quality control of the conjugate vaccines and vaccine intermediates must be validated. As may be required, proposals for the modification of manufacturing and control methods should also be submitted for approval to the NRA before they are implemented. Streptococcus pneumoniae is a Biological Safety Level (BSL) 2 pathogen and represents a particular hazard to health through infection by the respiratory route. The organism should be handled under appropriate conditions for this class of pathogen (27). Standard operating procedures need to be developed for dealing with emergencies arising from the accidental spillage, leakage or other dissemination of pneumococcal organisms. Personnel employed in the production and control facilities should be adequately trained and appropriate protective measures including vaccination with a pneumococcal vaccine licensed for use in adults should be

8 Page 8 implemented. Adherence to current Good Manufacturing Practices is important to the integrity of the product, to protect workers and to protect the environment. A.3 Production Control A.3.1 Control of Polysaccharide A Strains of Streptococcus pneumoniae The strains of S. pneumoniae used for preparing the polysaccharide should be agreed with the NRA. Each strain should have been shown to be capable of producing polysaccharide of the appropriate serotype. Each master seed lot should be identified by a record of its history, including the source from which it was obtained and the tests made to determine the characteristics of the strain. The cultures may be examined for the following characteristics: microscopically, stained smears from a culture should appear typical of S. pneumoniae; the organism should grow at 37 o C, but not at 25 o C, and should have characteristic smooth alpha haemolytic colonies; the ability to ferment inulin; the organism should be lysed in the bile solubility test and be sensitive to Optochin; a suspension of the culture should be agglutinated or give a positive Quellung reaction with the appropriate serotyping serum. Nuclear magnetic resonance spectroscopy (either 1 H or 13 C) is a suitable method for the confirmation of identity of purified polysaccharide. A Seed lot system The production of pneumococcal polysaccharide should be based on a working seed lot system. Cultures derived from the working seed lots shall have the same characteristics as the cultures of the strain from which the master seed lot was derived (A.3.1.1). If materials of animal origin are used in the medium for seed production, preservation of strain viability for freeze-drying or for frozen storage, then they should comply with the WHO Guidelines on Transmissible Spongiform Encephalopathies (28) and should be approved by the NRA. Manufacturers are encouraged to avoid wherever possible the use of materials of animal origin. A Culture media for the production of pneumococcal polysaccharide The liquid culture medium used for vaccine production should be free from ingredients that will form a precipitate upon purification of the capsular polysaccharide. If materials of animal origin are used then they should comply with the WHO Guidelines on Transmissible Spongiform Encephalopathies (28) and should be approved by the NRA. Manufacturers are encouraged to avoid wherever possible the use of materials of animal origin. A Single harvests Consistency of growth of S. pneumoniae should be demonstrated by monitoring growth rate, ph and the final yield of polysaccharide.

9 Page 9 A Control of bacterial purity Samples of the culture should be taken before killing and be examined for microbial contamination. The purity of the culture should be verified by suitable methods, which should include inoculation on to appropriate culture media, including plate media that do not support growth of S. pneumoniae. If any contamination is found, the culture or any product derived from it should be discarded. The killing process should also be adequately validated. A Purified polysaccharide Each lot of pneumococcal polysaccharide should be tested for identity, purity and molecular size. A number of approaches to determining polysaccharide identity and purity give complementary but incomplete information, so a combination of methods should be employed to provide all necessary data and should be agreed by the NRA. The purity limits given below are expressed with reference to the polysaccharide in its salt form (sodium or calcium), corrected for moisture. Variations in these specifications that may be appropriate if unusual salt forms are present should be agreed by the NRA. Generally, after killing the organism the culture is harvested and the polysaccharide isolated and purified by techniques such as fractional precipitation, chromatography, enzyme treatment and ultrafiltration. The polysaccharide is partially purified by fractional precipitation, washed and dried to a residual moisture content shown to favour the stability of the polysaccharide. s used for the purification of bulk polysaccharide should be approved by the NRA. Purified pneumococcal polysaccharide and, when necessary partially purified intermediates, are usually stored at or below -20 C to ensure stability. A Polysaccharide identity A test should be performed on the purified polysaccharide to verify its identity. In cases where other polysaccharides are produced on the same manufacturing site, the method should be validated to show that it distinguishes the desired polysaccharide from all other polysaccharides produced on that manufacturing site. A serological method such as countercurrent immunoelectrophoresis and/or nuclear magnetic resonance spectroscopy (either 1 H or 13 C) provide convenient methods for this purpose (29-31). In some cases, the identity of the polysaccharide can be deduced from its composition, if appropriate analytical methods are employed. A Polysaccharide composition The composition of the polysaccharide provides information on its purity, identity and the amount of specific impurities, such as pneumococcal C-polysaccharide, that are present. Analyses should be based on the dry weight of the polysaccharide. The composition of the polysaccharide can be defined in a number of ways depending on the methodology employed and the salt form present. The specifications used should be agreed by the NRA. Chemically, the composition of pneumococcal polysaccharides can be defined by the percentage of total nitrogen, phosphorus, uronic acid, hexosamine, methyl pentose and O-acetyl groups. These are usually determined by a combination of simple wet

10 Page 10 chemical tests with colorimetric read outs. Typical specifications are tabulated below (32). They may be adapted when other methods such as 1 H-NMR are used. Other methods, such as High Performance anion exchange chromatography (HPAEC) with electrochemical detection, with pulsed amperometric detection (HPAEC-PAD) applied to hydrolysates of the polysaccharide, may be used to define aspects of the quantitative composition of certain polysaccharide types, but the method should be validated for the purpose (33). 1 H nuclear magnetic resonance spectroscopy also provides a convenient approach to quantitatively define the composition of the purified polysaccharide if an internal reference compound is included (30, 31). The proportion of pneumococcal C polysaccharide may be determined by a combination of 1 H and 31 P nuclear magnetic resonance spectroscopy (34, 35) or HPAEC-PAD (36).

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12 Page 12 Table 2 Theoretical composition of pneumococcal polysaccharides * Serotype Total nitrogen Phosphorus a Uronic acid Hexosamines Methyl pentose O-acetyl groups (%) (range) (%) (range) (%) (%) (%) (%) (3.5 6) 0 (0 1.5) ( 45) ( 1.8) 2 0 (0 1) 0 (0 1.0) ( 15) ( 38) (0 1) 0 (0 1.0) ( 40) (4 6) 0 (0 1.5) ( 40) ( 10) (2.5 6) 0 (<2) ( 12) ( 20) ( 25) 0 6B 0 (0 2) 4.38 ( ) ( 15) 0 7F 2.28 ( ) 0 (0 1.0) ( 13) 3.5 (present) 8 0 (0 1) 0 (0 1.0) ( 25) N 3.09 ( ) 0 (0 1.0) ( 20) ( 28) 0 0 9V 1.44 (0.5 3) 0 (0 1.0) ( 15) ( 13) (present) 10A 1.12 ( ) 2.48 ( ) ( 12) A 0 (0 2.5) 3.25 ( ) ( 9) 12F 3.82 (3 5) 0 (0 1.0) ( 15) ( 25) ( 10) (1.5 4) 0 (0 1.0) ( 20) B 1.31 (1 3) 2.89 ( ) ( 15) (present) 17A 0 (0 1.5) 0 (0 3.5) ( 10) ( 20) 3.2 (present) 17F 0 (0 1.5) 2.93 (0 3.5) ( 20) 4.06 (present) 18C 0 (0 1) 3.05 ( ) ( 14) 4.24 (present) 19A 2.27 ( ) 5.04 ( ) ( 12) ( 20) 0 19F 2.27 ( ) 5.04 ( ) ( 12.5) ( 20) ( ) 0 ( ) ( 12) (present) 22F 0 (0 2) 0 (0 1.0) ( 15) ( 25) 4.22 (present) 23F 0 (0 1) 3.90 ( ) ( 37) 0 33F 0 (0 2) 0 (0 1.0) (present) * theoretical value with suggested range in parenthesis, based on published structures. These are calculated using broad definitions of the classes of sugars, so, for example hexosamine include 2-acetamido-2,6-dideoxyhexoses and 2-acetamido-2-deoxyuronic acids, methylpentose includes 2-acetamido-2,6-dideoxyhexoses and uronic acid includes 2-acetamido-2-deoxyuronic acids. It is not certain that such sugars would give an identical response in chemical tests used to determine the composition. The values are cited as equivalents of probably reference compounds used in such compositional tests. The values assume complete O-acetylation at each distinct site for O-acetylation, using published and unpublished data

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14 Page 14 A Moisture content If the purified polysaccharide is to be stored as a lyophilised powder the moisture content should be determined by suitable methods approved by the NRA and shown to be within agreed limits. A Protein impurity The protein content should be determined by the method of Lowry et al., using bovine serum albumin as a reference (37), or other suitable validated method. Sufficient polysaccharide should be assayed to detect 1% protein contamination accurately. Each lot of purified polysaccharide should typically contain not more than 3% by weight of protein. However, this will vary depending upon the serotype and an acceptable level of protein contamination should be agreed with the NRA. A Nucleic acid impurity Each lot of polysaccharide should contain not more than 2% by weight of nucleic acid as determined by ultraviolet spectroscopy, on the assumption that the absorbance of a 1 g/l nucleic acid solution contained in a cell of 1 cm path length at 260 nm is 20 (38) or by another validated method. Sufficient polysaccharide shall be assayed to detect 2% nucleic acid contamination accurately. A Pyrogen content The pyrogen content of the purified polysaccharide should be determined and shown to be within acceptable limits agreed by the NRA. A recognized pyrogenicity test can be performed in rabbits. Alternatively, the Limulus amoebocyte lysate test can be performed. A Molecular Size Distribution The molecular size of each lot of purified polysaccharide provides an indication of the manufacturing consistency. An acceptable level of consistency should be agreed with the NRA and can be established either by process validation or measurement on each lot. The distribution constant (K D ) can be determined by measuring the molecular size distribution of the polysaccharide at the main peak of the elution curve obtained by a suitable chromatographic method. The K D value and/or the mass distribution limits should be established. s such as: gel filtration through Sepharose CL-4B or CL-6B (or similar) in a 0.2 molar buffer using either a refractive index detector or colorimetric assay for the detection of the polysaccharide; and high performance size-exclusion chromatography (HPSEC) with refractive index detectors either alone or in combination with light scattering (e.g. Multiple Angle Laser Light Scattering, MALLS) are suitable for this purpose (31, 39). The methodology and column used should be validated to demonstrate sufficient resolution in the appropriate molecular weight range.

15 A Modified polysaccharide Page 15 Modified polysaccharide preparations may be partially depolymerised either before or during the chemical modification. The registered and several candidate pneumococcal conjugate vaccines use polysaccharides and oligosaccharide chains. A Chemical modification Several methods for the chemical modification of polysaccharides prior to conjugation may be satisfactory. The chosen method should be approved by the NRA. The current methods used are similar to those employed in the production of conjugate vaccines against Haemophilus influenzae type b. For example, polysaccharide may be oxidised with periodate and the periodate activated polysaccharide attached to free amino groups on the carrier protein by reductive amination. Alternatively, the polysaccharide can be randomly activated by cyanogen bromide, or a chemically similar reagent, and a bifunctional linker added, which then allows the polysaccharide to be attached to the carrier protein directly, or through a secondary linker. A Extent of modification of the polysaccharide The manufacture should demonstrate consistency of the degree of modification of the polysaccharide, either by an assay of each batch of the polysaccharide or by validation of the manufacturing process. Depending on the conjugation chemistry used, consistency in degree of polysaccharide activation may be determined as part of process validation or reflected by characteristics of vaccine lots shown to have adequate safety and immunogenicity in clinical trials. A Molecular size distribution The degree of size reduction of the polysaccharide will depend upon the manufacturing process. The average size distribution (degree of polymerization) of the modified polysaccharide should be determined by a suitable method and shown to be consistent. The molecular size distribution should be specified for each serotype, with appropriate limits for consistency, as the size may affect the reproducibility of the conjugation process. The molecular size may be determined by gel filtration on soft columns or by HPSEC on using refractive index alone, or in combination with laser light scattering (e.g. MALLS) (31, 39). An alternative method shown to correlate to molecular size distribution (e.g. measurement of viscosity) may be used to show consistency to size reduction of the PS. A.3.2 Control of the carrier protein A Microorganisms and culture media for production of carrier protein Microorganisms to be used for the production of the carrier protein should be grown in media free from substances likely to cause toxic or allergic reactions in humans. If any materials of animal origin are used in seed preparation, or preservation, or in production, they should comply with the WHO Guidelines on Transmissible Spongiform Encephalopathies (28) and should be approved by the NRA.

16 Page 16 Production should be based on a seed lot system with the strains identified by a record of their history and of all tests made periodically to verify strain characteristics. Consistency of growth of the microorganisms used should be demonstrated by monitoring the growth rate, ph and final yield of appropriate protein(s). A Characterization and purity of the carrier protein Potentially there are many proteins that could be used as carriers in pneumococcal conjugate vaccines. The principal characteristics of the carrier protein should be that it is safe and, in the conjugate, elicits a T-cell dependent immune response against the polysaccharide. Test methods used to characterize such proteins, to ensure that they are non-toxic and to determine their purity and concentration, should be approved by the NRA. Proteins and purification methods that might be used include: 1. Tetanus or diptheria toxoid. This must satisfy the relevant requirements published by WHO (40) and be of high purity (41). 2. Diphtheria CRM 197 protein. This is a non-toxic mutant of diphtheria toxin, isolated from cultures of Corynebacterium diphtheriae C7 /β197 (42). Protein of purity should be greater than 90% as determined by an appropriate method. When produced in the same facility as diphtheria toxin, methods must be in place to distinguish the CRM 197 protein from the active toxin. 3. Protein D derived from non-typeable Haemophilus influenzae. The routine release should include tests to confirm identity and purity of the protein as approved by the NRA, supplemented by additional data to characterize the protein. The protein carrier should also be characterized. The identity may be determined serologically. Physico-chemical methods that may be used to characterize protein include SDS-PAGE, isoelectric focusing, HPLC, amino acid analysis, amino acid sequencing, circular dichroism, fluorescence spectroscopy, peptide mapping and mass spectrometry as appropriate (31). A.3.3 Control of monovalent bulk conjugates There are a number of possible conjugation methods that might be used for vaccine manufacture; all involve multi-step processes. Both the method and the control procedures used to ensure the reproducibility, stability, and safety of the conjugate should be established for licensing. The derivatization and conjugation process should be monitored by analysis for unique reaction products or by other suitable means. The conditions used in the conjugation chemistry may affect the structure of the polysaccharide chain by causing the loss of labile substituents. Unless the combination of tests used to characterize the bulk monovalent conjugate provide this information, an explicit identity test on the polysaccharide present should be performed. Residual activated functional groups potentially capable of reacting in vivo may be present following the conjugation process. The manufacturing process should be validated to show that the activated functional groups do not remain at the conclusion of the manufacturing process or inferior to a limit approved by the NRA. After the conjugate has been purified, the tests described below are usually performed on nonadsorbed conjugate bulks. Alternatively, they may be performed on adsorbed monovalent

17 Page 17 conjugate bulks, e.g; in case individual conjugate bulks are adsorbed to adjuvant prior to final formulation of the vaccine. The tests are critical for assuring lot-to-lot consistency. A Identity A test should be performed on the monovalent bulk to verify its identity. The method should be validated to show that it distinguishes the desired monovalent material from all other polysaccharides and conjugates produced on that manufacturing site. A Residual reagents The conjugate purification procedures should remove residual reagents used for conjugation and capping. The removal of reagents and reaction by-products such as cyanide, 1-ethyl-3,3-(3- dimethylaminopropyl)-carbodiimide (EDAC) and others, depending on the conjugation chemistry, should be confirmed by suitable tests or by validation of the purification process. The residuals are process-specific and can be quantified by use of colorimetric and chromatographic assays. Techniques such as NMR spectroscopy and hyphenated techniques such as LC-MS may also be applied. A Polysaccharide-protein ratio and conjugation markers For each batch of the bulk conjugate of each serotype the ratio of polysaccharide to carrier protein should be determined as a marker of the consistency of the conjugation chemistry. For each conjugate, the ratio should be within the range approved for that particular conjugate by the NRA and should be consistent with vaccine shown to be effective in clinical trials. Typically for pneumococcal conjugate vaccines the ratio is in the range of 0.3 to 3.0 but varies with the serotype. The ratio can be determined either by independent measurement of the amounts of protein and polysaccharide present, or by methods which give a direct measure of the ratio. s include 1 H nuclear magnetic resonance spectroscopy or the use of HPSEC with dual monitoring (eg. refractive index and UV, for total material and protein content respectively). If the chemistry of conjugation results in the creation of a unique linkage marker (eg. a unique amino acid), each batch of the bulk conjugate of that serotype should be assessed to quantify the extent of degree of substitution of the carrier protein by covalent reaction of the pneumococcal polysaccharide with the carrier protein. The structural complexity and structural differences between the pneumococcal serotypes are such that in most cases a simple conjugation marker will not be able to be identified. A Capping markers Each batch should be shown to be free of activated functional groups on either the chemically modified polysaccharide or carrier protein. Alternatively, the product of the capping reaction can be monitored or the capping reaction can be validated to show removal of unreacted functional groups. Validation of the manufacturing process during vaccine development can eliminate the need to perform this analysis for routine control.

18 Page 18 A3.3.5 Conjugated and unbound (free) polysaccharide Only the pneumococcal polysaccharide that is covalently bound to the carrier protein, i.e. conjugated polysaccharide, is immunologically important for clinical protection. Each batch of conjugate should be tested for unbound or free polysaccharide in order to establish consistency of production and to ensure that the amount present in the purified bulk is within the limits agreed by the NRA based on lots shown to be clinically safe and efficacious. s that have been used to separate unbound polysaccharide prior to assay, and potentially applicable to pneumococcal conjugates, include hydrophobic chromatography, acid precipitation, precipitation with carrier protein-specific antibodies, gel filtration and ultrafiltration. The amount of unbound polysaccharide can be determined by specific chemical or immunological tests, or by HPAEC after hydrolysis. A Protein content The protein content of the conjugate should be determined by means of an appropriate validated assay and comply with limits for the particular product. Each batch should be tested for conjugated and unbound protein. If possible, the unconjugated protein should also be measured. Appropriate methods for the determination of conjugated and unconjugated protein include HPLC or capillary electrophoresis. A Molecular size distribution The molecular size of the polysaccharide-protein conjugate is an important parameter in establishing consistency of production and in studying stability during storage. The relative molecular size of the polysaccharide-protein conjugate should be determined for each bulk, using a gel matrix appropriate to the size of the conjugate. The method should be validated with an emphasis on specificity to distinguish the polysaccharide-protein conjugate from other components that may be present, e.g. unbound protein or polysaccharide. The size distribution specifications will be vaccine specific and should be consistent with lots shown to be immunogenic in clinical trials. A Sterility Typically the size may be examined by gel filtration on Sepharose CL-2B, or by HPSEC on an appropriate column. Since the saccharide-protein ratio is an average value, characterization of this ratio over the size distribution (e.g. by dual monitoring of the column eluent) can be used to provide further proof of manufacturing consistency (43). The bulk purified conjugate should be tested for bacterial and mycotic sterility in accordance with the requirements of Part A, sections 5.1 and 5.2, of the revised Requirements for Biological Substances (44) or by a method approved by the NRA. If a preservative has been added to the product, appropriate measures should be taken to prevent it from interfering with the test. A Specific toxicity of carrier protein

19 Page 19 The bulk conjugate should be tested for the absence of specific toxicity of the carrier protein where appropriate (e.g. when tetanus or diphtheria toxoids have been used). Absence of specific toxicity of the carrier protein may also be assessed through validation of the production process. A Endotoxin content To ensure an acceptable level of endotoxin in the final product, the endotoxin content of the monovalent bulk may be determined and shown to be within acceptable limits agreed by the NRA. A.3.4 Final bulk A Preparation To formulate the final bulk, monovalent conjugate bulks may be mixed together and an adjuvant, a preservative and/or stabilizer is added before final dilution. Alternatively, the monovalent conjugate bulks may also be adsorbed to adjuvant individually before mixing them to formulate the final vaccine. A Sterility Each final bulk should be tested for bacterial and mycotic sterility as indicated in section A.3.5 Filling and containers The recommendations concerning filling and containers given in Good Manufacturing Practices for Biological Products should be applied (26). A.3.6 Control tests on final product A Identity An identity test should be performed which demonstrates that all of the intended pneumococcal polysaccharide serotypes and carrier protein(s) are present in the final product, unless this test has been performed on the final bulk. A Sterility A serological test, using antibodies specific for the purified polysaccharide may be used. The contents of final containers should be tested for bacterial and mycotic sterility as indicated in section A Pneumococcal polysaccharide content The amount of each pneumococcal polysaccharide in the final containers should be determined, and shown to be within the specifications agreed by the NRA. The conjugate vaccines produced by different manufacturers differ in formulation. A quantitative assay for each the pneumococcal polysaccharides in the final container

20 Page 20 should be carried out. The assays used are likely to be product specific and might include chromatographic or serological methods. Immunological assays such as rate nephelometry (45) or ELISA inhibition may be used. Assessment of the content of each serotype in the final vaccine may be difficult and may require complex methodologies not available to the national control laboratories (NCLs). Therefore, in the event that testing is performed in the framework of lot release by NCLs, measurement of the total polysaccharide content could be authorized. A Residual moisture If the vaccine is freeze-dried, the average moisture content should be determined by methods accepted by the NRA. Values should be within limits of the preparations shown to be adequately stable in the stability studies of the vaccine. The test should be performed on 1 vial per 1000 up to a maximum of 10 vials but on no less than 5 vials taken at random from throughout the final lot. The average residual moisture content should generally be no greater than 2.5% and no vial should be found to have a residual moisture content of 3% or greater. A Endotoxin content The vaccine in the final container should be tested for endotoxin content by a Limulus amoebocyte lysate test (LAL). Endotoxin content or pyrogenic activity should be consistent with levels found to be acceptable in vaccine lots used in clinical trials and approved by the NRA. A Adjuvant content If an adjuvant has been added to the vaccine, its content should be determined by a method approved by the NRA. The amount and nature of the adjuvant should be agreed with the NRA. If aluminium compounds are used as adjuvants, the amount of aluminium should not exceed 1.25 mg per single human dose. A Preservative content The manufacturer has a choice of possible preservatives. Consideration should be given to the stability of the chosen preservative and possible interactions between the vaccine components and the preservative. If a preservative has been added to the vaccine, the content of preservative should be determined by a method approved by the NRA. The amount of preservative in the vaccine dose should be shown not to have any deleterious effect on the antigen nor impair the safety of the product in humans. The preservative and its concentration should be approved by the NRA. A General safety test (Innocuity) The requirement to test lots of pneumococcal conjugate vaccine for unexpected toxicity (abnormal toxicity) should be agreed with the NRA. Such a test may be omitted for routine lot release once consistency of production has been well established to the satisfaction of the NRA and when Good Manufacturing Practice is in place.

21 Page 21 A ph If the vaccine is a liquid preparation, the ph of each final lot should be tested and shown to be within the range of values found for vaccine lots shown to be safe and effective in the clinical trials and in stability studies. For a lyophilized preparation, the ph should be measured after reconstitution with the appropriate diluent. A Inspection of final containers Each container in each final lot should be inspected visually (manually or with automatic inspection systems), and those showing abnormalities such as improper sealing, lack of integrity and, if applicable, clumping or the presence of particles should be discarded. A.4 Records The recommendations in Section 8 of Good Manufacturing Practices for Biological Products should be applied (26). A.5 Retained samples The recommendations in Section 9.5 of Good Manufacturing Practices for Biological Products should be applied (26). A.6 Labelling The recommendations in Section 7 of Good Manufacturing Practices for Biological Products should be applied with the addition of the following (26). The label on the carton or the leaflet accompanying the container should indicate: the pneumococcal serotype and carrier protein present in each single human dose; the amount of each conjugate present in a single human dose; the temperature recommended during storage and transport; if the vaccine is freeze-dried, that after its reconstitution it should be used immediately unless data have been provided to the licensing authority that it may be stored for a limited time; the volume and nature of the diluent to be added in order to reconstitute a freeze-dried vaccine, specifying that the diluent should be supplied by the manufacturer and approved by the NRA. A.7 Distribution and transport The recommendations in Section 8 of Good Manufacturing practices for Biological Products should be applied (26). A.8 Stability, storage and expiry date A.8.1 Stability testing